C.W.G.M. Löwik

Effects of experimental conditions on the release of 45calcium from prelabeled fetal mouse long bones.

Embryonic/neonatal bones in culture are commonly used for the study of osteoclastic resorption in vitro. For this purpose, the release of 45calcium (45Ca) from prelabeled bones is measured as an index of resorption. We studied 45Ca release from two types of long bone explants after different preparation methods: 17-day-old fetal mouse radii/ulnae with and without cartilage ends (intact radii/ulnae and shafts, respectively), and intact 18-day old metacarpals/metatarsals. In addition, we examined the effect of different culture conditions, such as cultures performed under the surface of the medium or at the interphase of medium and air, on 45Ca release and histology. When intact radii/ulnae were cultured under the surface of the medium, there was always a significant amount (10%) of net basal 45Ca release (corrected for physicochemical exchange) that was not due to osteoclastic resorption, as it could not be suppressed by inhibitors of resorption even at high concentrations. Moreover, histologically TRAcP-positive cells were almost absent after culture and the bone marrow/stromal cells in the center of the bone appeared necrotic, possibly due to a lack of oxygen. Under these culture conditions, osteoclasts could survive in shafts as well as in PTH-stimulated intact radii/ulnae, but a constant amount of 10% 45Ca, not due to resorption, was still released in the medium. When these explants were cultured at the interphase of medium and air, basal and stimulated 45Ca release originated from osteoclastic resorption. In contrast, in 18-day-old fetal mouse metacarpals/metatarsals, the experimental conditions applied did not affect 45Ca release, which was always due to resorption of the explants by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vivo Molecular Bioluminescence Imaging: New Tools and Applications

in vivo bioluminescence imaging (BLi) is an optical molecular imaging technique used to visualize molecular and cellular processes in health and diseases and to follow the fate of cells with high sensitivity using luciferase-based gene reporters. The high sensitivity of this technique arises from efficient photon production, followed by the reaction between luciferase enzymes and luciferin substrates. Novel discoveries and developments of luciferase reporters, substrates, and gene-editing techniques, and emerging fields of applications, promise a new era of deeper and more sensitive molecular imaging.

Photodynamic-Immune Checkpoint Therapy Eradicates Local and Distant Tumors by CD8+ T Cells

Local tumor destruction by photodynamic therapy induces potent antitumor T-cell responses, which can be enhanced by systemic CTLA-4 blockade to eliminate local and distant tumors in mice. This provides an alternative approach to treating advanced multifocal cancer. Photodynamic therapy (PDT) is a clinically applied tumor ablation method that reduces tumor burden and may induce T-cell responses, providing a therapeutic option for mutated tumors. In this study, we applied PDT in two mouse tumor models and assessed its effect on outgrowth of PDT-treated and distant untreated tumors. PDT of established tumors resulted in complete tumor eradication in most mice, which were then protected against tumor rechallenge. Correspondingly, the therapeutic effect was abrogated upon systemic depletion of CD8+ T cells, indicating PDT-induced tumor antigen cross-presentation and T-cell activation. In a double-tumor model, PDT of primary tumors induced enhanced infiltration of untreated distant tumors by CD8+ T cells, which significantly delayed their outgrowth. Combination therapy of PDT and CTLA-4–blocking antibodies significantly improved therapeutic efficacy and survival of double-tumor–bearing mice. These results show that local tumor ablation by PDT induces CD8+ T-cell responses crucial for systemic tumor eradication, which can be further enhanced by combination with immune checkpoint blockade. This combination of two clinically applied therapies may be a treatment strategy for advanced cancer without previous knowledge of tumor-specific antigens. Cancer Immunol Res; 5(10); 832–8. ©2017 AACR.

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging.

PurposeRecently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1xa0cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomographyxa0(SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA).ProceduresThe necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo.ResultsUsing optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis.ConclusionsThe radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosisxa0in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy.

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death

Purpose Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with 111Indium-chloride (111In-Cl3) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. Materials and methods Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9u2009Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40u2009h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [111In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography–computed tomography (SPECT–CT) at the same time points. Results In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. Conclusion HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients.

Composites of hydroxyapatite and bisphosphonate: properties and alveolar bone response

A composite was made by adsorption of the bisphosphonate [(3-dimethylamino-1-hydroxypropylidene)-1,1-bisphosphonate; dimethyl-APD] into an hydroxyapatite (HA) tube. Adsorbed dimethyl-APD did not change the bulk properties of the HA tube but the surface properties were altered. The amount of 0.1 mmol/L dimethyl-APD adsorbed into the HA tube was 0.78 ( ± 0.20) μg after 4 weeks. The composite tube of HA and dimethyl-APD placed after extraction of teeth in the premolar regions of dogs were biocompatible, stable and bonded strongly and intimately to the alveolar bone. Although there was no bone resorption around the composite tubes, no conclusion can be drawn yet from this study as far as local inhibition of alveolar bone resorption is concerned.

Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter

Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging. However, sensitive longitudinal imaging of transplanted stem cells in deep tissue like the brain remains challenging not only due to low light penetration but because of other factors such as low or inferior expression levels of optical reporters in stem cells and stem cell death after transplantation. Here we describe an optimized imaging protocol for sensitive long-term monitoring of bone marrow-derived human mesenchymal stem cells (hMSCs) expressing a novel bioluminescent/near infrared fluorescent (NIRF) fusion reporter transplanted in mouse brain cortex. Lentivirus expressing the luc2-iRFP720 reporter, a fusion between luc2 codon-optimized firefly luciferase (luc2) and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were analyzed for their fluorescent and bioluminescent emission and checked for their differentiation potential. In vivo experiments were performed by transplanting decreasing amounts of luc2-iRFP720 expressing hMSCs in mouse brain, followed by fluorescence and bioluminescence imaging (BLI) starting 1 wk after cell injection when the blood–brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luc2 substrate performances, that is, D-luciferin (D-Luc; 25 μM/kg or 943 μM/kg) or CycLuc1 (25 μM/kg). Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential toward adipocytes, chondrocytes, and osteocytes, suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1 × 105 cells using both fluorescence and BLI. The highest bioluminescent signals (∼1 × 107 photons per second) were achieved 15 min after the injection of D-Luc (943 μM/kg). This allowed us to monitor as low as 1 × 105 hMSCs for the subsequent 7 wk without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.

Fluorescence-Guided Surgery: A Promising Approach for Future Oncologic Surgery

Imaging is currently used for cancer detection, staging, and treatment evaluation. However, during surgery, surgeons rely mostly on visual inspection and palpation. Fluorescence imaging could provide the surgeon with necessary real-time information and could therefore revolutionize surgical oncology. This chapter gives an extensive overview of the current status of preclinical and clinical fluorescence-guided surgery. First, the basic principles of optical imaging and the specificity of fluorescence imaging will be presented. Secondly, fluorescence-guided sentinel lymph node (SLN) mapping will be discussed. The SLN is of vital importance for cancer staging and consequently influences the choice of therapy and survival rates. We will discuss preclinical and especially clinical work as many trials appraise the use of fluorescence in SLN mapping. In the final part, we will focus on specific targeting of the primary tumor by fluorescent agents that take aim of the alterations in cell physiology that denote cancer.

Detecting tumour-positive resection margins after oral cancer surgery by spraying a fluorescent tracer activated by gamma-glutamyltranspeptidase

OBJECTIVESnTumour-positive resection margins are a major problem during oral cancer surgery. gGlu-HMRG is a tracer that becomes fluorescent upon activation by gamma-glutamyltranspeptidase (GGT). This study aims to investigate the combination of gGlu-HMRG and a clinical fluorescence imaging system for the detection of tumour-positive resection margins.nnnMATERIALS AND METHODSnThe preclinical Maestro and clinical Artemis imaging systems were compared in vitro and ex vivo with cultured human head and neck cancer cells (OSC19, GGT-positive; and FaDu, GGT negative) and tumour-bearing nude mice. Subsequently, frozen sections of normal and oral cancer tissues were ex vivo sprayed with gGlu-HMRG to determine the sensitivity and specificity. Finally, resection margins of patients with suspected oral cancer were ex vivo sprayed with gGlu-HMRG to detect tumour-positive resection margins.nnnRESULTSnBoth systems could be used to detect gGlu-HMRG activation in vitro and ex vivo in GGT positive cancer cells. Sensitivity and specificity of gGlu-HMRG and the Artemis on frozen tissue samples was 80% and 87%, respectively. Seven patients undergoing surgery for suspected oral cancer were included. In three patients fluorescence was observed at the resection margin. Those margins were either tumour-positive or within 1u202fmm of tumour. The margins of the other patients were clear (≥8u202fmm).nnnCONCLUSIONnThis study demonstrates the feasibility to detect tumour-positive resection margins with gGlu-HMRG and a clinical fluorescence imaging system. Applying this technique would enable intraoperative screening of the entire resection margin and allow direct re-resection in case of tumour-positivity.

Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer

OBJECTIVESnEstablishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis.nnnMATERIAL AND METHODSnTo determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated.nnnRESULTSnAll human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150μg) and 0.34nmol (50μg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes.nnnCONCLUSIONnThis study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.